Methods for modulating D1-D2 dopamine receptor coupling and function

ABSTRACT

The present invention provides for prevention and/or treatment of neurological or neuropsychiatric disorders involving abnormal D1-D2 dopamine receptor coupling and/or activation. Methods and agents are provided for modulating dopamine receptor function arising from D1-D2 coupling and/or activation. Agents of the present invention include fragments of D2 receptor or D1 receptor that can disrupt D1-D2 coupling.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/997,813 filed Dec.13, 2010, now U.S. Pat. No. 8,420,775 issued Apr. 16, 2013, which is theU.S. National Phase of PCT Appln. No. PCT/CA2009/000829 filed Jun. 12,2009, now expired, which claims the benefit of U.S. provisionalapplication Ser. No. 61/060,948 filed Jun. 12, 2008, the disclosures ofwhich are incorporated in their entirety by reference herein.

SEQUENCE LISTING

The text file dated Jan. 16, 2015 and titled “SequenceListing.txt” ofsize 15 KB, filed herewith, is hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to psychiatric diseases or disorders, andmore particularly, compositions and methods for modulating theinteraction and function of D1-D2 dopamine receptors. Such compositionsand methods are useful for prevention and/or treatment of psychiatricdiseases or disorders, particularly depression, including majordepressive illness and depression in bipolar disorder, and psychiatricconditions that require treatment with medication, includingschizophrenia, psychosis in bipolar disorder, and stimulant drugintoxication.

BACKGROUND OF THE INVENTION

Depression:

Depression is a mood disorder characterized by depressed mood; feelingsof worthlessness, helplessness or hopelessness; a loss of interest orpleasure; changes in appetite; change in sleeping pattern; fatigue,thoughts of death, inability to concentrate or make decisions. Accordingto Statistics Canada's 2002 Mental Health and Well-being Survey⁴, 12.2%of all Canadians will experience depression within their lifetime, while4.8% of Canadians had reported symptoms for major depression.Furthermore, the economical impact of depression is tremendous due tocosts in both productivity and health care. In Canada, between 62% and76% of short-term disability episodes due to mental disorders wereattributed to depression⁵. Work-related productivity losses due todepression have been estimated to be $4.5 billion⁶. Thus, the prevalenceof depression makes this disorder a very important health issue inCanada and abroad.

Once diagnosed, depression can be treated by different therapiesincluding medication, psychotherapy and in more severe cases, withelectroconvulsive therapy. The first line of treatment is often throughantidepressant medication, sometimes in conjunction with psychotherapy.Antidepressants consist of the classical tricyclic antidepressants(TCA), selective serotonin reuptake inhibitors (SSRI), noradrenaline andserotonin reuptake inhibitor (NSRI), as well as monoamine oxidaseinhibitors (MAOI). All antidepressants have acute effects on synapticlevels of neurotransmitters in the brain⁷⁻⁹. The classical TCAs arepredominantly noradrenaline and serotonin reuptake inhibitors, similarto NSRIs. The SSRI drugs are more selective serotonin transporterinhibitors, while MAOI block enzymes that are involved in the breakdownof these neurotransmitters.

Dopamine (DA), acting through DA receptors, exerts a major role inregulating neuronal motor control, cognition, event prediction, emotionand pleasure/reward¹⁰⁻¹⁵, all of which are affected in depression. Thecontribution of DA in depression becomes evident when taking intoaccount the major dopaminergic pathways in the mammalian brain: (a) themesostriatal system consisting of dopaminergic neurons from thesubstantia nigra (SNc) innervating the striatum; (b) the mesolimbicsystem in which dopaminergic neurons from the ventral tegmental area(VTA) project into the hippocampus, nucleus accumbens (NAc) andamygdala; (c) the mesocortical system where DA neurons mostly from theVTA project into the cortical regions of the brain including theprefrontal cortex (PFx). Most of these regions have been implicated indepression. Furthermore, numerous studies support the hypothesis ofdecreased dopaminergic signalling in depression including reports that:(1) the severity of major depression correlates highly with patientresponse to amphetamine, a drug that facilitates increased synaptic DAlevels through multiple mechanisms¹⁶, while another study has showndecreased levels of homovanillic acid, a major DA metabolite, in the CSFof depression patients¹⁷; (2) animals experiencing learned helplessness,a behavioural paradigm that recapitulates some of the symptoms ofdepression, have been shown to exhibit DA depletion in the striatum,which can be mitigated by pretreatments with DA agonists¹⁸⁻¹⁹; (3) motoreffects induced by DA receptor agonists are increased after chronictreatment with antidepressants or electroconvulsive therapy²⁰ suggestingreduced dopaminergic neurotransmission in depression; (4) in forced-swimtests, DA agonists have been shown to inhibit immobility, an indicationof antidepressant activity, while DA receptor antagonists have beenshown to inhibit the effects of antidepressants²¹⁻³²; (5) DAT inhibitorsnomifensine and bupropion have been shown to be effectiveantidepressants^(21, 33-34) and (6) clinical studies have alsodocumented cases where DA receptor agonists have been effective intreating depression³⁵⁻³⁹. Furthermore, there is some evidence fromneuroimaging studies that dopamine D2 receptor (D2R) are elevated in thestriatum of depressed patients⁴⁰⁻⁴⁴.

Another brain pathway implicated in depression is thehypothalamic-pituatary-adrenal (HPA) axis. The HPA axis is involved instress reaction and ultimately leads to increased secretion ofglucocorticoids from the adrenal cortex. Although glucocorticoids haveeffects on the hippocampus it has also been shown to facilitate DAtransmission in NAc⁴⁵. In addition, frequent bouts of stress withintermittent exposure to glucocorticoids sensitize the mesolimbic DAsystem⁴⁶. While the hippocampus and frontal cortex are undoubtedlyinvolved in certain aspects of depression, symptoms of anhedonia, lackof motivation and motor deficits implicate other regions of the brainincluding the dorsal and ventral striatum, which are rich indopaminergic neurons. Moreover, serotonergic activity has an impact onDA neurotransmission. Studies have shown that stimulation of 5HT_(1A)receptors can stimulate DA release in PFx and NAc but inhibit DA releasein the dorsal striatum⁴⁷. Other studies have shown that activation ofserotonergic raphe neurons reduces activity of dopaminergic neurons inVTA (not SNc) and inhibit locomotion, exploratory behaviour⁴⁸⁻⁴⁹.

Schizophrenia:

Schizophrenia is a severe chronic and debilitating mental disorder thatstrikes in youth and affects not only patients but their families andcare-givers⁵⁰⁻⁵¹. Approximately 2.2 million American adults haveschizophrenia in a given year. Clinical symptoms of schizophreniainclude delusions, hallucinations, disorganized thinking, and cognitivedysfunction that are divided into two major groups: positive andnegative symptoms⁵².

Accumulated evidence suggests that the positive symptoms result fromhyperdopaminergia involving dopamine D2 receptors in the limbicstriatum, while the negative/cognitive symptoms arise from ahypodopaminergic function mediated by dopamine D1 receptors in theprefrontal cortex⁵². Despite decades of intensive research, currentantipsychotic medications are still limited to the blockade of D2receptor function that generally alleviate positive symptoms with onlylimited impact on cognitive and negative symptoms and can induce seriousside effects including extrapyramidal side effects (EPS). Patientscontinue to experience significant disability and functional impairmentthat limits their integration in society. The unavailability ofeffective medications with both D1 agonism and D2 antagonism is mainlydue to the unknown therapeutic target, a pathway through which bothinhibition of D2 receptor and activation of D1 receptor function can beachieved.

The dopamine hypothesis of schizophrenia, in its original formulationaddressed mainly positive symptoms⁵³⁻⁵⁴. Early pharmacotherapy forschizophrenia involved the use of reserpine, which blocks dopaminerelease from presynaptic terminals, and/or the use of antipsychotics⁵⁵.Moreover, the most compelling evidence for the involvement of dopaminereceptors in schizophrenia comes from the fact that most antipsychotics,including atypical antipsychotics, show a dose-dependent threshold of D2receptor occupancy for their therapeutic effects⁵⁵. The efficacy of bothreserpine and antipsychotics in treating schizophrenia stronglyimplicate the involvement of dopamine in this neuropsychiatric disorder.More recent versions of this theory suggests that while the positivesymptoms result from hyperdopaminergia in the limbic striatum, thenegative/cognitive symptoms arise from a hypodopaminergic function inthe prefrontal cortex (PFC)⁵⁶⁻⁵⁷. A significant body of literature lendssupport to this theory. PET & SPECT studies have shown evidence ofincreased dopamine synthesis, release, and levels in thesubcortical/limbic regions⁵⁸ while functional imaging studies havedemonstrated hypofunction in the prefrontal cortex at baseline and whileperforming cognitive tasks⁵⁹. More recent studies have focused on thedopamine D1 system in the PFC, as it is the predominant dopaminereceptor sub-type in the PFC⁶⁰⁻⁶¹, and show a decrease in receptornumber which correlates with executive dysfunction⁶² and a compensatoryup-regulation which correlates with working-memory dysfunction⁶³. Theseclinical observations are well supported by preclinical evidence—PFC D1receptor modulation changes the ‘memory fields’ of prefrontal neuronssubserving working memory⁶⁴⁻⁶⁵ and D1 agonist administration improvesworking memory performance in both aging and dopamine deficientmonkeys⁶⁶ (as it does in aging human subjects)⁶⁷⁻⁶⁸.

Overview of DA Receptors:

In mammals, five distinct genes, termed D1/D5 for D1-like receptors andD2/D3/D4 for D2-like receptors, encode DA receptors. These receptorsbelong to a super-family of single polypeptide seven trans-membrane (TM)domain receptors that exert their biological effects via intracellularG-protein coupled signaling cascades¹. D1 and D5 receptorspreferentially couple to Gs proteins stimulating the activity ofadenylate cyclase and PKA dependent pathways. D2 receptors display amore complex pattern of signal transduction primarily due to theircoupling to subtype specific members of the Gi/Go protein family.

D2 receptors are known to stimulate a number of signal transductionpathways including the inhibition of adenylate cyclase activity, PIturnover, potentiation of arachidonic acid release, inwardly rectifyingK⁺ and Ca²⁺ channels and mitogen activated protein kinases². Moreover,studies have shown that protein interactions can play a large role in DAreceptor function. For instance, the D2R has been shown to physicallyinteract with Par-4. Interestingly, Par-4 mutant mice, which are unableto interact with D2R, exhibit depression-like behaviour³.

Dopamine D1-D2 Receptor Link:

While numerous studies have indicated a synergy between D1 and D2receptors, an interesting study by Seeman et al (1989)⁶⁹ provided thefirst evidence of a pharmacological link between D1 and D2 receptors.Briefly, it was shown that dopamine could lower the density of D2receptors labeled by [³H] raclopride and that the addition of thespecific D1 receptor antagonist, SCH-23390, prevented this reduction,suggesting a functional link between D1 and D2 receptors. Interestingly,this pharmacological D1-D2 link was absent or reduced in post-mortembrain tissues of approximately half of the schizophrenia populationtested. However, it remains unclear if the absence of the D1-D2 link isdue to an inherent dissociation between the D1 and D2 receptor thatoccurs in schizophrenia or is a result of antipsychotic drug treatment.Furthermore, several studies using a combination of D1 and D2 specificagonists and/or antagonists have shown that co-activation of D1-D2receptors are required for long-term depression, anandamide-mediatedmemory consolidation and potentiation of immediate early gene response,suggesting a potential functional interaction between the D1R andD2R⁷⁰⁻⁷⁶.

Recent evidence indicates that D1 and D2 receptors form a proteincomplex, and co-activation of D1 and D2 receptors results in an increaseof intracellular calcium levels via a signaling pathway not activated byeither receptor alone, confirming the functional link observed betweenD1 and D2 receptors⁷⁷⁻⁷⁸. Furthermore, it has been shown that D1 and D2receptors are co-expressed in neurons of the rat striatum, providing abasis for a functional interaction⁷⁹⁻⁸⁰.

Despite years of research in the field of mental health, there continuesto be a need for new and improved medicines for treating psychiatricdiseases and disorders, including depression, schizophrenia andpsychotic symptoms thereof. The present inventors have accordinglysought to identify new diagnostic and chemotherapeutic methods in thisarea by investigating the functional association between D1 and D2classes of DA receptors.

SUMMARY OF THE INVENTION

The present invention accordingly relates to compositions and methodsfor prevention and/or treatment of diseases and disorders involvingabnormal DA receptor association and functionality. More particularly,the present invention relates to methods of modulating the interactionand functionality of D1-D2 receptors, as well as compounds useful insuch methods. The invention also relates to methods of diagnosis ofdiseases and disorders caused by abnormal D1-D2 receptor association andfunctionality.

The present invention provides compounds, compositions and methods formodulating the interaction of D1-D2 receptors. Furthermore, the presentinvention provides methods for preventing and/or treating diseasesinvolving abnormal levels of D1-D2 interaction and/or functionality.

According to the present invention there is provided a method formodulating dopamine (DA) receptor function in a mammal in need of suchtreatment comprising administering a therapeutically effective amount ofan agent that disrupts D1-D2 coupling in the mammal. In a preferredembodiment, which is not meant to be limiting in any manner, the agentis a peptide or polypeptide.

In an embodiment, the agent is an antibody that binds to an amino acidsequence that is at least 80% identical to the sequence of any one ofthe sequences selected from D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2)(SEQ ID NO:2), D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4). In afurther embodiment, the amino acid sequence is identical to the sequenceof D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-L)(SEQ ID NO:3), or D1_(CT) (SEQ ID NO:4).

In a further embodiment, the agent is a nucleic acid encoding apolypeptide of between about 3 and about 140 amino acids and comprisingan amino acid sequence that is at least 80% identical to the sequence ofany one of the sequences selected from D2_(IL3-29) (SEQ ID NO:1),D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQID NO:4). The polypeptide, in some embodiments, may be identical to asequence of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2),D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4).

In further embodiments, the agent may be a polypeptide of between about3 and about 140 amino acids comprising an amino acid sequence that isbetween about 80% and about 100% identical to any one of the sequencesselected from the group consisting of D2_(IL3-29) (SEQ ID NO:1),D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQID NO:4). The polypeptide, in such embodiments, may be identical to asequence of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2),D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4).

The above method can be for preventing and/or treating a diseaseselected from the group consisting of depression, including majordepressive illness and depression in bipolar disorder, and psychiatricconditions that require treatment with antipsychotic medication,including schizophrenia, psychosis in bipolar disorder, and stimulantdrug intoxication.

As a further aspect of the invention, there is provided a polypeptide ofbetween about 3 and about 140 amino acids comprising an amino acidsequence that is at between about 80% and about 100% identical to thesequence of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2),(SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4).

The polypeptide may comprise an amino acid sequence that is betweenabout 80% and 100% identical to a sequence selected from the groupconsisting of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2),D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4). More specifically,the polypeptide may comprise an amino acid sequence that is identical toD2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-L) (SEQID NO:3), or D1_(CT) (SEQ ID NO:4).

In a yet further aspect of the invention, there is provides a nucleicacid encoding a polypeptide of between 3 and 140 amino acids comprisingan amino acid sequence that is between about 80% identical and 100%identical to the sequence of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2)(SEQ ID NO:2), D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4).

The nucleic acid may encode a polypeptide having an amino acid sequencethat is between 80% and 100% identical to a sequence selected from thegroup consisting of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ IDNO:2), D2_(IL3-L) (SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4). In a furtherembodiment, the nucleic acid encodes a polypeptide that is identical toa sequence selected from D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQID NO:2), D2_(IL3-L)(SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4).

A protein transduction domain may be fused or linked to any smallmolecule chemical compound, polypeptide, nucleic acid, or combinationthereof, used in the context of the present invention. In certainnon-limiting representative examples, the protein transduction domain isselected from the group consisting of TAT, and SynB1/3Cit. Also, otherproteins, non-protein carriers or other macromolecules may be attachedor conjugated to the compound, polypeptide, nucleic acid describedabove.

The present invention also provides a kit comprising any component orcombination of components as mentioned or described throughout.

As described herein, a direct interaction between D1 and D2 receptors,has been identified, and the present invention provides agents thatspecifically disrupt this interaction. Furthermore, the presentinvention provides methods for identifying agents that disrupt theinteraction between the D1 and D2 receptors.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings, in which:

FIG. 1A shows the results of a Western blot analysis in which CO-IP ofD1 receptor by D2 receptor (antibody) in solubilized rat striataltissue;

FIG. 1B shows the results of a Western blot analysis in which D2receptor is specifically pulled down by GST-D1_(CT) in detergentextracts of rat striatum, but not GST-D1_(IL3) (third intracellularloop) or GST alone;

FIG. 1C shows the results of a Western blot analysis in which D1receptor was specifically pulled down by GST-D2_(IL3-L) in detergentextracts of rat striatum, but not GST-D2_(IL3-S) or GST alone;

FIG. 1D shows the results of a Western blot analysis in which D1receptor was specifically pulled down by GST-D2_(IL3-L) andGST-D2_(IL3-29) in detergent extracts of rat striatum, but notGST-D2_(CT), GST-D2_(IL3-S) or GST alone;

FIG. 1E shows the results of a Western blot analysis in which D1receptor was pulled down by GST-D2_(IL3-29-2), but not GST-D2_(IL3-29-1)or GST alone;

FIG. 1F shows the results of a Western blot analysis of an in vitrobinding assay, wherein a S³⁵-D1 tail probe specifically bound withGST-D2_(IL3-29-2) fragment, but not with GST-D2_(IL3-29-1) or GST alone;

FIG. 1G shows the results of a Western blot analysis of competitiveCO-IP in rat striatum. The ability of D1 receptor toco-immunoprecipitate with D2 receptor is inhibited by the addition ofGST-D2_(IL3-29-2) in a concentration dependent manner;

FIG. 2A shows the results of a time course of 2 μM Fluo-4AMfluorescence, corresponding to increases in intracellular Ca²⁺ levels inHEK-293T cells co-expressing D1 and D2 receptors, following simultaneousactivation by both 10 μM SKF81297 and quinpirole. Fluorescence valueswere monitored using a PerkinElmer multiwell plated fluorometer,collected at 3-s intervals for 150 s. (A.F.U.: arbitrary fluorescenceunits). The curve shown is representative of three replicatemeasurements performed;

FIG. 2B shows the measurement of fluorescence, corresponding to theintracellular Ca²⁺ levels in HEK-293T cells co-expressing D1 and D2receptors treated with 10 μM SKF81297, 10 μM quinpirole or both. **Significant vs. control group (p<0.01). Significant vs. SKF+Quin group(#, p<0.05; ##, p<0.01). Data is representative of three replicatemeasurements performed. Data was analyzed by one-way ANOVA, followed byNewman-Keuls test;

FIG. 3A shows fluorescence measurements for HEK-293T cells co-expressingD1 and D2 receptors and pretreated with 10 μM raclopride or SCH23390.The cells were stimulated with both 10 μM SKF81297 and 10 μM quinpirole.Significant vs. control group (*, p<0.05; **, p<0.01). ### Significantvs. SKF+Quin group (p<0.001) Data is representative of three replicatemeasurements performed. Data was analyzed by one-way ANOVA, followed byNewman-Keuls test;

FIG. 3B shows fluorescence measurements for HEK-293T cells co-expressingD1 and D2 receptors and pretreated with 10 μM U73122 (PLC inhibitor).The cells were stimulated with 10 μM SKF81297 and 10 μM quinpirole.Significant vs. control group (**, p<0.01). ### Significant vs. SKF+Quingroup (p<0.001). Data is representative of three replicate measurementsperformed. Data was analyzed by one-way ANOVA, followed by Newman-Keulstest;

FIG. 3C shows fluorescence measurements for HEK-293T cells co-expressingD1 and D2 receptors in the presence of D1_(CT) or D5_(CT) mini-genes andstimulated with a 10 μM concentration of both SKF81297 and quinpirole.*, **Significantly from control group (p<0.05; p<0.01). # Significantlyfrom SKF+Quin group (p<0.05). Data is representative of three replicatemeasurements performed. Data was analyzed by one-way ANOVA, followed byNewman-Keuls test;

FIG. 4 shows the results of characterization of the D1-D2 interaction inpost-mortem brain. Striatal post-mortem brain samples (control,schizophrenia, bipolar and depression; 15 samples in each group),obtained from the Stanley Foundation, were incubated with anti-D2receptor antibodies for coimmunoprecipitation experiments. Precipitatedproteins were subject to SDS-PAGE; immunoblotted with D1 antibody.Co-immunoprecipitation of D1 by the D2 antibody is significantlyincreased in depression brains compared to controls. Data were analyzedby one-way ANOVA followed by post-hoc SNK tests (* P<0.05, n=15);

FIG. 5 (A) illustrates the results of an analysis of D1-D2 interaction,in which it is shown that chronic antidepressant treatment results in adecrease in D1-D2 interaction. Rats were treated with imipramine (IMI,10 mg/kg/day) or saline for 14 days. On the 14th day, rats weresacrificed, brains quickly extracted and striata were dissected forbiochemical analysis. Striata were solubilized and used incoimmunoprecipitation experiments to examine the coprecipitation of theD1 receptor with the D2 receptor. Rats subjected to chronic imipramine(IMI) exhibited a decrease in the D1-D2 interaction, as quantified fromWestern blots of co-immunoprecipitation samples (*, p<0.05, t-test,n=5);

FIG. 5 (B) illustrates the results of quantification of Western blotsfrom the control (Con) and IMI samples revealed no significantdifferences in D1 receptor levels (n=5). Tubulin was used as loadingcontrols;

FIG. 5 (C) illustrates the results of quantification of Western blotsfrom the control (Con) and IMI samples revealed no significantdifferences in D2 receptor levels (n=5). Tubulin was used as loadingcontrols;

FIG. 6 (A) illustrates the results of a study of rats subjected tolearned helplessness (LH), in which an increase in the D1-D2 interactionis shown. Rats that were subjected to LH were compared against controlrats. Striata were solubilized and used in co-immunoprecipitationexperiments to examine the co-precipitation of the D1 receptor with theD2 receptor. Rats exhibited an increase in the D1-D2 interaction afterLH, as quantified from Western blots of co-immunoprecipitation samples(**, p<0.01, n=5, t-test).

FIG. 6(B) illustrates the results where prefrontal cortex (PFC) weresolubilized and used in co-immunoprecipitation experiments to examinethe co-precipitation of the D1 receptor with the D2 receptor. Ratsexhibited an increase in the D1-D2 interaction after LH, as quantifiedfrom Western blots of co-immunoprecipitation samples (*, p<0.05, n=5,t-test);

FIG. 7 illustrates the results of a study of rats subjected to chronicmild stress (CMS), in which an increase in the D1-D2 interaction isshown. Rats that were subjected to CMS were compared against controlrats. Prefrontal cortex were solubilized and used inco-immunoprecipitation experiments to examine the co-precipitation ofthe D1 receptor with the D2 receptor. Rats exhibited an increase in theD1-D2 interaction after CMS, as quantified from Western blots ofco-immunoprecipitation samples (*, p<0.05, n=5, t-test).

FIG. 8 (A) illustrates the results of a study of rats subjected toforced swim tests (FST), in which an increase in the D1-D2 interactionis shown. Rats that were subjected to forced swim tests were comparedagainst control rats for changes in D1 and D2 receptors. Rat striatawere solubilized and used in co-immunoprecipitation experiments toexamine the co-precipitation of the D1 receptor with the D2 receptor.Rats exhibited an increase in the D1-D2 interaction 3 hours or 3 daysafter FST trials, as quantified from Western blots ofco-immunoprecipitation samples (*, p<0.05, n=4, ANOVA post hoc SNK);

FIG. 8 (B) illustrates the results of quantification of Western blotsfrom the control (Con) and FST samples revealed no significantdifferences in D1 receptor levels. Tubulin was used as loading controls;

FIG. 8 (C) illustrates the results of quantification of Western blotsfrom the control (Con) and FST samples revealed no significantdifferences in D2 receptor levels (n=4). Tubulin was used as loadingcontrols;

FIG. 9 shows Western blots illustrating a significant decrease in D1-D2receptor complex formation in rat with chronic antipsychotic treatment(bottom panel) (by pump 0.25 mg/kg/day for 2 weeks), while directimmunoprecipitated D2 receptors remain unchanged (top panel);

FIG. 10 illustrates the results of a study in which disruption of D1-D2interaction leads to behavior changes in rats subjected to forced swimtests. Intra-PFC administration of TAT-D2L_(IL3-29-2), but not TATalone, decreased the frequencies of rat immobility and increased thefrequencies of rat swimming and climbing behaviors in a 5 min forcedswimming test. Each value is the mean.+−.S.E.M. for a group of 6 rats.Data were analyzed by one-way analysis of variance (ANOVA).

FIG. 11 shows the protein sequence of human D2R-L, The I₂₅₆-V₂₇₀ regionshown to be important for D1-D2 binding is underlined.

FIG. 12 shows the DNA sequence encoding the human D2R-L.

FIG. 13 shows the protein sequence of human D1R. The D1_(CR) (A₃₃₂-T₄₄₆)is shown in bold, while the third intracellular loop D1_(IL3)(R₂₁₉-K₂₇₂) is underlined in the D1 sequence.

FIG. 14 shows the DNA sequence encoding human DIR.

DETAILED DESCRIPTION

The following description is of a preferred embodiment.

The present invention provides a method for modulating dopamine (DA)receptor function, partially as a result of identifying a directinteraction between D1 and D2 receptors (see Examples). Agents thatspecifically disrupt the D1-D2 receptor interaction, and methods foridentifying agents that disrupt this interaction are accordinglyprovided. Formation of the D1-D2 complex has been shown herein to occurat elevated levels in individuals suffering from depression. Moreover,disruption of the D1-D2 interaction and complex formation is shown toreduce or alleviate symptoms of depression using animal models ofdepression. Accordingly, modulating DA receptor function throughdisruption of the D1-D2 complex can be effective for preventing and/ortreating a variety of neurological diseases and disorders, for example,but not limited to, depression, including major depressive illness anddepression in bipolar disorder, schizophrenia, psychotic symptoms ofschizophrenia, and other psychiatric conditions that require treatmentwith antipsychotic medication, such as psychosis in bipolar disorder,and stimulant drug intoxication.

By disrupting D1-D2 coupling, an agent may for instance inhibit bindingor otherwise prevent association or interaction between the D1 and D2receptor.

The method for modulating DA receptor function may involve administeringthe D1-D2 disrupting agent in a therapeutically effective amount. Suchamount may vary depending upon the disease or disorder to be treated, aswell as other pharmacological factors known to those skilled in the art.

By “agent” or “agents” it is meant any small molecule chemical compound,polypeptide, nucleic acid or any combination thereof that can modulateDA receptor function. By “modulate DA receptor function” is meant analtering of function, for instance by inhibition of DA activation and/orsignaling function, by disrupting D1-D2 coupling. A polypeptide may beof any length unless otherwise specified and includes, for example andwithout limitation, antibodies, enzymes, receptors, transporters, D2receptor, D1 receptor fragment or derivative. A fragment is anypolypeptide or nucleic acid that is shorter than its correspondingnaturally occurring polypeptide or nucleic acid, respectively. Aderivative is any polypeptide or nucleic acid that is altered withrespect to a reference polypeptide or nucleic acid, respectively, andincludes, for example fragments or mutants.

Accordingly, in a preferred embodiment, the present invention provides apolypeptide of less than 140 amino acids comprising an amino acidsequence that is at least 80% identical to the sequence of D2_(IL3-29)(SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-L) (SEQ ID NO:3),and D1_(CT) (SEQ ID NO:4), or a fragment of any one thereof. In apreferred embodiment, the polypeptide is between about 4 and about 140amino acids, for example, but not limited to 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 90 or 100 amino acids. In an alternate embodiment, thepolypeptide is between about 3 and 100 amino acids, for example, but notlimited to 5 and 100 amino acids, 7 and 100 amino acids, 9 and 100 aminoacids, 11 and 100 amino acids, 13 and 100 amino acids, or 15 and about140 amino acids, for example. However, it is to be understood that thesize of the peptide may be defined by a range of any two of the valueslisted above or any values therein between. Also, in an alternateembodiment, which is not meant to be limiting in any manner, the presentinvention contemplates polypeptides as defined above which comprisesmore than 140 amino acids.

It is to be understood that the polypeptide as described above does notconsist of the full amino acid sequence of any naturally occurring D1 orD2 receptor, or any naturally occurring allelic variant thereof.

The sequences of D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-)(SEQ ID NO:3),and D1_(CT) (SEQ ID NO:4) are as follows, with specific reference to thenumbering of the full length amino acid sequences of D1 and D2 shown inFIGS. 11 and 13:

D2 Peptides: D2_(IL3-29) G₂₄₂-V₂₇₀ (SEQ ID NO: 1)GNCTHPEDMKLCTVIMKSNGSFPVNRRRV D2L_(IL3-29-2) I₂₅₆-V₂₇₀ (SEQ ID NO: 2)IMKSNGSFPVNRRRV D2_(IL3-L) K₂₁₁-Q₃₇₃ (SEQ ID NO: 3)KIYIVLRRRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKT MSRRKLSQQKEKKATQD1 Peptides: D1_(CT) A₃₃₂-T₄₄₆ (SEQ ID NO: 4)AFNADFRKAFSTLLGCYRLCPATNNAIETVSINNNGAAMFSSHHEPRGSISKECNLVYLIPHAVGSSEDLKKEEAAGIARPLEKLSPALSVILDYDTD VSLEKIQPITQNGQHPT

The present invention also contemplates polypeptides having an aminoacid sequence that comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to the amino sequences described above. Further, thepolypeptides may be defined as comprising a range of sequence identitydefined by any two of the values listed above.

In an embodiment of the present invention, which is not meant to belimiting, there is provided a polypeptide from D2L_(IL3-29-2) comprisingor consisting of the amino acid sequence IMK, MKS, KSN, SNG, NGS, GSF,SFP, FPV, PVN, VNR, NRR, RRR, or RRV. Further, the polypeptide maycomprise or consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 aminoacids including one or more sequential amino acids attached at the Nterminus, C terminus or both of any of the sequences provided above thatare found in a naturally occurring D2 receptor or any allelic variantthereof.

In a further embodiment of the present invention, there is provided apolypeptide comprising or consisting of the amino acid sequence fromD1_(CT) comprising or consisting of AFN, FNA, NAD, ADF, DFR, FRK, RKA,KAF, AFS, FST, STL, TLL, LLG, LGC, GCY, CYR, YRL, RLC, LCP, CPA, PAT,ATN, TNN, NNA, NAI, AIE, IET, ETV, TVS, VSI, SIN, INN, NNN, NNG, NGA,GAA, AAM, AMF, MFS, FSS, SSH, SHH, HHE, HEP, EPR, PRG, RGS, GSI, SIS,ISK, SKE, KEC, ECN, CNL, NLV, LVY, VYL, YLI, LIP, IPH, PHA, HAV, AVG,VGS, GSS, SSE, SED, EDL, DLK, LKK, KKE, KEE, EEA, EAA, AAG, AGI, GIA,IAR, ARP, RPL, PLE, LEK, EKL, KLS, LSP, SPA, PAL, ALS, LSV, SVI, VIL,ILD, LDY, DYD, YDT, DTD, TDV, DVS, VSL, SLE, LEK, EKI, KIQ, IQP, QPI,PIT, ITQ, TQN, QNG, NGQ, GQH, QHP, and HPT. Further, the polypeptide maycomprise or consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 aminoacids including one or more sequential amino acids attached at the Nterminus, C terminus or both of any of the sequences provided above thatare found in a naturally occurring D1 receptor or any allelic variantthereof.

The present invention also provides a nucleic acid encoding polypeptidesas defined above. For example, but not wishing to be limiting in anymanner, the present invention contemplates a nucleic acid encoding apolypeptide of between about 3 and less than 140 amino acids, forexample, but not limited to between 10 and 135 amino acids, between 10and 100 amino acids, between 15 and 109 amino acids or between 15 and100 amino acids and that encodes an amino acid sequence that is at least80% identical to the sequence of D2L_(IL3-29-2) (SEQ ID NO:2),D2_(IL3-)(SEQ ID NO:3), and D1_(CT) (SEQ ID NO:4). In an alternateembodiment, the present invention contemplates nucleic acids ornucleotide sequences as described above but that encode more than 140amino acids. Such nucleic acids may be derived from the amino acidsequences above, or from the corresponding full length nucleic acidsequences of the D1 and/or D2 receptor coding sequences shown in FIGS.12 and 14.

By “percent identical” or “percent identity”, it is meant one or morethan one nucleic acid or amino acid sequence that is substantiallyidentical to a coding sequence or amino acid sequence of peptides thatcan disrupt D1-D2 coupling. By “substantially identical” is meant anynucleotide sequence with similarity to the genetic sequence of a nucleicacid of the invention, or a fragment or a derivative thereof. The term“substantially identical” can also be used to describe similarity ofpolypeptide sequences. For example, nucleotide sequences or polypeptidesequences that are at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 98%or 99% identical to the D1 or D2 receptor coding sequence, or theencoded polypeptide, respectively, or fragments or derivatives thereof,and still retain ability to affect D1-D2 coupling are contemplated.

To determine whether a nucleic acid exhibits identity with the sequencespresented herein, oligonucleotide alignment algorithms may be used, forexample, but not limited to a BLAST (GenBank URL:www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program:blastn; Database: nr; Expect 10; filter: default; Alignment: pairwise;Query genetic Codes: Standard(1)), BLAST2 (EMBL URL:http://www.embl-heidelberg.de/Services/index.html using defaultparameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect:10,cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), orFASTA, search, using default parameters. Polypeptide alignmentalgorithms are also available, for example, without limitation, BLAST 2Sequences (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html, using defaultparameters Program: blastp; Matrix: BLOSUM62; Open gap (11) andextension gap (1) penalties; gap x_dropoff: 50; Expect 10; Word size: 3;filter: default).

An alternative indication that two nucleic acid sequences aresubstantially identical is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Hybridization to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. for at least 1 hour (see Ausubel, et al. (eds), 1989,Current Protocols in Molecular Biology, Vol. 1, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).Alternatively, hybridization to filter-bound sequences under stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. for at least 1hour (see Ausubel, et al. (eds), 1989, supra). Hybridization conditionsmay be modified in accordance with known methods depending on thesequence of interest (see Tijssen, 1993, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, New York).Generally, but not wishing to be limiting, stringent conditions areselected to be about 5° C. lower than the thermal melting point for thespecific sequence at a defined ionic strength and pH.

By protein transduction domain it is meant a sequence of nucleic acidsthat encode a polypeptide, or a sequence of amino acids comprising thepolypeptide, wherein the polypeptide facilitates localization to aparticular site, for example a cell or the like, or it may facilitatetransport across a membrane or lipid bilayer. The polypeptides andnucleic acids of the present invention may be fused to a proteintransduction domain to facilitate transit across lipid bilayers ormembranes. Also, the present invention contemplates that the polypeptideas described herein may be linked or attached to a support, amacromolecule, a non-protein carrier or a protein carrier optionally viaa spacer compound, molecule or the like.

Many polypeptides and nucleic acids do not efficiently cross the lipidbilayer of the plasma membrane, and therefore enter into cells at a lowrate. However, there are certain naturally occurring polypeptides thatcan transit across membranes independent of any specific transporter.Antennapedia (Drosophila), TAT (HIV) and VP22 (Herpes) are examples ofsuch polypeptides. Fragments of these and other polypeptides have beenshown to retain the capacity to transit across lipid membranes in areceptor-independent fashion. These fragments, termed proteintransduction domains, are generally 10 to 27 amino acids in length,possess multiple positive charges, and in several cases have beenpredicted to be ampipathic. Polypeptides and nucleic acids that arenormally inefficient or incapable of crossing a lipid bilayer, can bemade to transit the bilayer by being fused to a protein transductiondomain.

U.S. Publication 2002/0142299 (which is incorporated herein byreference) describes a fusion of TAT with human beta-glucuronidase. Thisfusion protein readily transits into various cell types both in vitroand in vivo. Furthermore, TAT fusion proteins have been observed tocross the blood-brain-barrier. Frankel et al. (U.S. Pat. No. 5,804,604,U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No.5,670,617, and U.S. Pat. No. 5,652,122; which are incorporated herein byreference) have also demonstrated transport of a protein(beta-galactosidase or horseradish peroxidase) into a cell by fusing theprotein with amino acids 49-57 of TAT.

PCT publication WO01/15511 (which is incorporated herein by reference)discloses a method for developing protein transduction domains using aphage display library. The method comprises incubating a target cellwith a peptide display library and isolating internalized peptides fromthe cytoplasm and nuclei of the cells and identifying the peptides. Themethod further comprised linking the identified peptides to a proteinand incubating the peptide-protein complex with a target cell todetermine whether uptake is facilitated. Using this method a proteintransduction domain for any cell or tissue type may be developed. USPublication 2004/0209797 (which is incorporated herein by reference)shows that reverse isomers of several of the peptides identified by theabove can also function as protein transduction domains.

PCT Publication WO99/07728 (which is incorporated herein by reference)describes linearization of protegrin and tachyplesin, naturallyoccurring as a hairpin type structure held by disulphide bridges.Irreversible reduction of disulphide bridges generated peptides thatcould readily transit cell membranes, alone or fused to other biologicalmolecules. US Publication 2003/0186890 (which is incorporated herein byreference) describes derivatives of protegrin and tachyplesin that weretermed SynB1, SynB2, SynB3, etc. These SynB peptides were furtheroptimized for mean hydrophobicity per residue, helical hydrophobicmoment (amphipathicity), or beta hydrophobic moment. Various optimizedamphipathic SynB analog peptides were shown to facilitate transfer ofdoxorubicin across cell membranes. Further, doxorubicin linked to a SynBanalog was observed to penetrate the blood-brain-barrier at 20 times therate of doxorubicin alone.

The protein transduction domains described in the preceding paragraphsare only a few examples of the protein transduction domains availablefor facilitating membrane transit of small molecules, polypeptides ornucleic acids. Other examples are transportan, W/R, AlkCWK18, DipaLytic,MGP, or RWR. Still many other examples will be recognized by personsskilled in the art

A protein transduction domain and an agent of the present invention maybe placed together in sufficient proximity and maintained together for asufficient time to allow the protein transduction domain to influencepharmaceutical product performance of the agent. Contemplatedassociations of protein transduction domain and agent include, forexample and without limitation: non-covalent associations such aselectrostatic interactions, hydrogen bonding, ionic bonds or complexes,Van der Waals bonds; covalent linkages such as conventional methods ofcross-linking; linkages that are activated, in vitro and/or in vivo byelectromagnetic radiation; any covalent bond such as a peptide bond; anybiochemical interaction known to protein biochemists, such asbiotin/streptavidin, nickel/Histidine,glutathione/glutathione-S-transferase, or antigen/antibody; physicalassociations within matrix structures or encapsulating systems; etc.

The present invention provides an agent that may be any small moleculechemical compound, polypeptide, nucleic acid, or any combination thereofthat can modulate DA receptor functionality through disruption of D1-D2coupling. Accordingly, the present invention provides a polypeptide ofabout 3 to less than about 140 amino acids, preferably 7 to 109 aminoacids, more preferably 15 to 100 amino acids and comprising an aminoacid sequence that is at least 80% identical, for example, but notlimited to 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe sequence of D2_(IL3-29) (SEQ ID NO:1), D2L_(IL3-29-2) (SEQ ID NO:2),D2_(IL3-L) (SEQ ID NO:3), or D1_(CT) (SEQ ID NO:4). The presentinvention also provides a nucleic acid encoding a polypeptide of about 3to less than about 140 amino acids, preferably about 10 to about 109amino acids, more preferably about 15 to about 100 amino acids andcomprising an amino acid sequence that is at least 80% identical, forexample, but not limited to 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to the sequence of D2_(IL3-29) (SEQ ID NO:1),D2L_(IL3-29-2) (SEQ ID NO:2), D2_(IL3-L) (SEQ ID NO:3), or D1_(CT) (SEQID NO:4). The polypeptide or nucleic acid may optionally be fused to aprotein transduction domain.

A polypeptide of the invention can be synthesized in vitro or deliveredto a cell in vivo by any conventional method. As a representativeexample of an in vitro method, the polypeptide may be chemicallysynthesized in vitro, or may be enzymatically synthesized in vitro in asuitable biological expression system, such as without limitation, wheatgerm extract or rabbit reticulocyte lysate. As a representative exampleof an in vivo method, a DNA, RNA, or DNA/RNA hybrid molecule comprisinga nucleotide sequence encoding a polypeptide of the invention isintroduced into an animal, and the nucleotide sequence is expressedwithin a cell of an animal.

The nucleotide sequence may be operably linked to regulatory elements inorder to achieve preferential expression at desired times or in desiredcell or tissue types. Furthermore, as will be known to one of skill inthe art, other nucleotide sequences including, without limitation, 5′untranslated region, 3′ untranslated regions, cap structure, poly Atail, translational initiators, sequences encoding signalling ortargeting peptides, translational enhancers, transcriptional enhancers,translational terminators, transcriptional terminators, transcriptionalpromoters, may be operably linked with the nucleotide sequence encodinga polypeptide (see as a representative examples “Genes VII”, Lewin, B.Oxford University Press (2000) or “Molecular Cloning: A LaboratoryManual”, Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition(2001)). A nucleotide sequence encoding a polypeptide or a fusionpolypeptide comprising a polypeptide agent and a protein transductiondomain may be incorporated into a suitable vector. Vectors may becommercialy obtained from companies such as Stratagene or InVitrogen.Vectors can also be individually constructed or modified using standardmolecular biology techniques, as outlined, for example, in Sambrook etal. (Cold Spring Harbor Laboratory, 3rd edition (2001)). A vector maycontain any number of nucleotide sequences encoding desired elementsthat may be operably linked to a nucleotide sequence encoding apolypeptide or fusion polypeptide comprising a protein transductiondomain. Such nucleotide sequences encoding desired elements, include,but are not limited to, transcriptional promoters, transcriptionalenhancers, transcriptional terminators, translational initiators,translational, terminators, ribosome binding sites, 5′ untranslatedregion, 3′ untranslated regions, cap structure, poly A tail, origin ofreplication, detectable markers, afffinity tags, signal or targetpeptide, Persons skilled in the art will recognize that the selectionand/or construction of a suitable factor may depend upon severalfactors, including, without limitation, the size of the nucleic acid tobe incorporated into the vector, the type of transcriptional andtranslational control elements desired, the level of expression desired,copy number desired, whether chromosomal integration is desired, thetype of selection process that is desired, or the host cell or the hostrange that is intended to be transformed.

The DNA, RNA, or DNA/RNA hybrid molecule may be introducedintracellularly, extracellularly into a cavity, interstitial space, intothe circulation of an organism, orally, or by any other standard routeof introduction for therapeutic molecules and/or pharmaceuticalcompositions. Standard physical methods of introducing nucleic acidsinclude, but are not limited to, injection of a solution comprising RNA,DNA, or RNA/DNA hybrids, bombardment by particles covered by the nucleicacid, bathing a cell or organism in a solution of the nucleic acid, orelectroporation of cell membranes in the presence of the nucleic acid.

A nucleic acid may be introduced into suitable eukaryotic cells ex vivoand the cells harboring the nucleic acid can then be inserted into adesired location in an animal. A nucleic acid can also be used totransform prokaryotic cells, and the transformed prokaryotic cells canbe introduced into an animal, for example, through an oral route. Thoseskilled in the art will recognize that a nucleic acid may be constructedin such a fashion that the transformed prokaryotic cells can express andsecrete a polypeptide of the invention. Preferably, the prokaryotic cellis part of the animal's endogenous intestinal microflora. With regardsto human examples of endogenous microflora are, without wishing to belimiting, Lactobacillus acidophillus, Streptococcus thermophilus, andBifidobacterium bifidum. A nucleic acid may also be inserted into aviral vector and packaged into viral particles for efficient deliveryand expression.

Dosage Forms

An agent of the present invention, for example, D1 or D2 polypeptides ornucleic acids encoding these polypeptides or antibodies or smallmolecules capable of disrupting D1-D2 coupling, may be formulated intoany convenient dosage form. The dosage form may comprise, but is notlimited to an oral dosage form wherein the agent is dissolved, suspendedor the like in a suitable excipient such as but not limited to water. Inaddition, the agent may be formulated into a dosage form that could beapplied topically or could be administered by inhaler, or by injectioneither subcutaneously, into organs, or into circulation. An injectabledosage form may include other carriers that may function to enhance theactivity of the agent. Any suitable carrier known in the art may beused. Also, the agent may be formulated for use in the production of amedicament. Many methods for the productions of dosage forms,medicaments, or pharmaceutical compositions are well known in the artand can be readily applied to the present invention by persons skilledin the art.

Combination therapy with agents of the present invention or other agentsuseful for preventing and/or treating neurological diseases or disordersis contemplated. With regards to combination therapy suitable dosageforms again include capsules, tablets, and the like, preferably for oraladministration, although any dosage form, for any route ofadministration is contemplated. Combination therapy can be administeredas separate entities, e.g. two tablets or other forms, each containingone agent, or may be administered as a single dosage form containingboth drugs, or concomitant use.

In case of oral administration of two or more different agents, thesingle dose can be, but is not limited to a capsule, tablet, or oralsolution, and it may also contain inactive component(s) that isnecessary to form the single delivery system.

Combination therapy medications of the present invention may beadministered by any desired route, for example without limitation,administration can be transdermal (patch), buccal, sublingual, topical,nasal, parenteral (subcutaneous, intramuscular, intravenous,intradermal,), rectal, vaginal, administration. Various combinations ofcontrolled release/rapid release are also contemplated.

Treatment

The methods and compounds of the present invention are useful forpreventing and/or treating diseases that are characterized by abnormallevels of D1-D2 interaction or complex formation. The following are somenon-limiting examples of such diseases: depression, including majordepressive illness and depression in bipolar disorder, and psychiatricconditions that require treatment with antipsychotic medication,including schizophrenia, psychosis in bipolar disorder, stimulant drugintoxication, delirium, Tourette's syndrome, anxiety or a combinationthereof.

Neurological, Neuropsychiatric Diseases

Depression is characterized by profound sadness, pronounced changes insleep, appetite, and energy. Recurrent thoughts of death or suicide,persistent physical symptoms that do not respond to treatment, such asheadaches, digestive disorders, and chronic pain are some symptoms ofmajor depression. Major depression is a unipolar depression, whilebipolar disorder (manic depression) involves both depression and mania.Early identification and treatment of depression is required to minimizerisk of suicide and self-inflicted injury. The method and compounds ofthe present invention are useful in decreasing D1-D2 coupling and may beused for preventing and/or treating depression.

Accordingly the present invention provides methods for modulating DAfunctionality by disrupting D1-D2 coupling in a mammal. Any mammalincluding, without limitation, human, rat, cow, pig, dog, or mouse, maybe treated with the agents and methods of the present invention.

The present invention will be further illustrated in the followingexamples.

EXPERIMENTS Experiment 1 D1-D2 Receptor Complex is Facilitated by theD1R Carboxyl Tail (CT) and the Third Intracellular Loop (IL3) of D2R

To confirm previous reports of a D1-D2 receptor complex we used ratstriata in co-immunoprecipitation (co-IP) experiments. As shown in FIG.1A, the D1R was able to co-precipitate with the D2R, confirming thepresence of a D1-D2 receptor complex. In an attempt to define thestructural basis for the observed D1-D2 coupling, we carried outaffinity purification using GST (glutathione-S-transferase)-fusionproteins encoding the D1_(CT) and the third intracellular loop(D1_(IL3)), since both D1_(CT): A₃₃₂-T₄₄₆ and D1_(IL3): R₂₁₉-K₂₇₂contain putative consensus sequences for receptor phosphorylation,desensitization and potential binding sites for various proteinsimportant for signalling (e.g. G proteins, NMDA receptor NR1, NR2Asubunits)⁸¹⁻⁸². As shown in FIG. 1B, GST-D1_(CT), but not GST-D1_(IL3)or GST alone, precipitated solubilized striatal D2R as illustrated bythe D2 antibody immunolabeled Western blot, indicating that the D1R caninteract with D2R through the D1_(CT) region. To locate the interactingsite on D2R, GST-fusion proteins encoding IL3 from both the D2 short(D2S) and D2 Long (D2L) (GST-D2_(IL3-L): K₂₁₁-Q₃₇₃,GST-D2_(IL3-S):K₂₁₁-Q₃₁₅) were used in affinity purification assays. Asshown in FIG. 1C, GST-D2_(IL3-L), but not GST-D2_(IL3-S) or GST alonewas able to pull-down D1 receptors. Since the D2L and D2S aredifferentiated by the additional 29 amino-acid within the thirdintracellular loop, the fact that only D2L but not D2S interacts withD1R made us suspect that this specific 29 amino-acid may contain theD1-D2 interacting site. The affinity “pull down” results revealed thatthe sequence encoded by the D2_(IL3-29) facilitates the interactionbetween D1 and D2 receptors since only the GST-D2_(IL3-L) andGST-D2_(IL3-29):G₂₄₂-V₂₇₀ but not GST-D2_(IL3-S), GST-D2_(CT):T₃₉₉-C₄₁₄or GST alone was able to pull-down D1 receptors (FIG. 1D). Furtherexperiments using fragments of D2_(IL3-29) show thatGST-D2L_(IL3-29-2):I₂₅₆-V₂₇₀, but not the GST-D2L_(IL3-29-1):G₂₄₂-V₂₅₅or GST, can successfully pull-down D1R from solubilized rat striatum(FIG. 1E). Furthermore, in vitro binding assay suggested directinteraction between D1R and D2R (FIG. 1F). These results provideevidence that the D1-D2 direct interaction is dependent on sequenceslocated in the I₂₅₆-V₂₇₀ region of D2R. Further, as shown in FIG. 1G,preincubation of GST-D2-IL3-29-2 inhibits the D1-D2 interaction asindexed by the co-immunoprecipitation in a concentration dependentmanner.

Experiment 2 Co-Activation of the D1-D2 Receptor Induces an Increase inIntracellular Ca²⁺ Mediated by Phospholipase C Activation

To investigate the functional implication of the D1-D2 coupling, weexamined the ability of this complex to promote Gq/11 signaling asindexed by changes in intracellular Ca²⁺ levels⁸³⁻⁸⁵. In HEK-293T cellsco-transfected with D1R and D2R we examined changes in intracellularCa²⁺ levels when co-treated with 10 μM SKF81297 and 10 μM quinpirole.Cells that were treated with both agonists exhibited a rapid andsignificant increase in intracellular Ca²⁺ levels that peaked 30 secondsafter addition of agonists (FIG. 2A). However, cells treated with eitherSKF81297 or quinpirole alone had no change in Ca²⁺ levels when comparedto nontreated control cells (FIG. 2B). The increase in Ca²⁺ levels couldbe blocked by pretreatment with either 10 μM raclopride (D2 antagonist)or 10 μM SCH23390 (D1 antagonist), as shown in FIG. 3A. Furthermore,this unique D1-D2 co-activation dependent signaling is mediated byphospholipase C (PLC) activation, since pretreatment with the PLCinhibitor U73122 (10 μM) inhibited the increase in Ca²⁺ levels inducedby co-activation of D1R and D2R (FIG. 3B). These data are in line withprevious studies showing that this signaling likely recruits Gq/11 uponco-activation of DA receptors, leading to downstream activation ofPLC⁸³⁻⁸⁵.

Experiment 3 Disruption of D1-D2 Coupling Abolished the D1-D2Co-Activation Induced Increases in Intracellular Ca²⁺

Our preliminary data has shown that coexpression of the GST-D1_(CT) isable to affinity pull down the D2R (FIG. 1B). Thus, to test whetherD1-D2 coupling is necessary for the D1-D2 co-activation inducedincreases in intracellular Ca²⁺, we examined changes in intracellularCa²⁺ levels when co-treated with 10 μM SKF81297 and 10 μM quinpirole inHEK-293T cells cotransfected with mini-genes encoding D1_(CT), D5_(CT)along with D1R and D2R. As shown in FIG. 3C, coexpression of the D1_(CT)mini-gene but not the D5_(CT) mini-gene abolished D1-D2 co-activationinduced increases in intracellular Ca²⁺ suggesting that the D1-D2coupling may be responsible for the observed increases in intracellularCa²⁺ induced by D1/D2 co-activation.

Experiment 4 D1-D2 Coupling is Upregulated in Post-Mortem Brain Tissueof Depression Patients

Both D1R and D2R have been implicated in the pathology of psychiatricdiseases such as schizophrenia. To test whether D1-D2 coupling isaltered in disease, we carried out co-immunoprecipitation experiments ina double-blind manner on 60 post-mortem brain striatum samples from theStanley Foundation, which includes 15 samples from each of four groups:control, schizophrenia, bipolar and severe depression. The four groupswere matched by age, sex, race, postmortem interval, pH, side of brain,and mRNA quality by the Stanley Foundation brain bank. The same amountof protein from each sample was incubated with anti-D2 receptor antibodyand protein A/G agarose. The precipitated proteins were divided equallyinto two groups before being subjected to SDS-PAGE and immunoblottedwith either D1 antibody or D2 antibody. Each Western blot included 3samples from each group and the intensity of each protein band wasquantified by densitometry (software: AIS from Imaging Research Inc).Each sample is presented as the percentage of the mean of three controlsamples on the same blot. As shown in FIG. 4A, theco-immunoprecipitation of D1 by the D2 receptor antibody wassignificantly enhanced in the depression post-mortem brain samplescompared to control brains. The levels of directly immunoprecipitated D2receptors were not significantly different between the control anddepression groups (data not shown). Therefore, the observed D1-D2coupling upregulation seen in the depression brain samples may be aprimary aspect of depression pathophysiology. However, we are aware thattwo issues may have influenced the results: (i) we do not have thepatient history of antidepressant usage, and (ii) that some of thedepression patients had history of drug abuse (5 out of 15) and/oralcohol abuse (10 out of 15), which may account for the large standarderror in our results. Given the complex nature of data involving humanbrain tissue, we have confirmed the D1-D2 coupling is enhanced in ratswith depressive-like behaviours induced by three differentuncontrollable stress paradigms.

Experiment 5 Chronic Antidepressant Treatment Leads to a Decrease in theD1-D2 Coupling

If the observed increases in the D1-D2 coupling in the postmortem braintissue of depression patients were indeed part of the pathologicalfoundation of depression, one would imagine that chronic antidepressanttreatment might correct such a change in the D1-D2 coupling. To testthis hypothesis, we injected rats subcutaneously with 10 mg/kg/day ofimipramine, a TCA, for a period of 14 days. On the 14th day, rats weresacrificed, striata dissected and were processed for co-IP assays andWestern blots. Co-immunoprecipitation experiments revealed that chronicantidepressant treatment led to a decrease in D1-D2 coupling (FIG. 5A).Furthermore, this decrease in the D1-D2 coupling could not be attributedto changes in receptor levels, since Western blots revealed nosignificant change in D1R and D2R levels when comparing chronicallytreated rats with control rats (FIG. 5B,C).

Experiment 6 D1-D2 Coupling is Up-Regulated in Animals withDepressive-Like Behaviours

This was tested by co-immunoprecipitation of the D1R by the D2R primaryantibody from solubilized proteins extracted from striatum, PFC of ratsfrom the two animal models with depressive-like behaviours compared tothe control groups. Briefly, anti-D2 antibody is incubated withsolubilized protein for 4 hours followed by the addition of protein A/Gbeads. Incubation with protein A/G beads continues overnight followed byhigh stringency washes. The immunoprecipitated proteins are eluted fromthe beads and subjected to SDS-PAGE for Western blot analysis. Controlexperiments without D2 antibody are carried out concurrently. Theco-immunoprecipitated proteins were immunoblotted with the D1, antibodyand the intensity of each protein band was quantified by densitometry.Each co-immunoprecipitation is in parallel with western blot analysis ofthe initial levels of solubilized protein and directlyimmunoprecipitated proteins. As shown in FIG. 6, 7, striata (LH model)and PFC from rats (LH model and CMS model) showed a significant increasein the D1-D2 coupling compared to control rats (n=5 p<0.05).Furthermore, there was no significant change in D1R or D2R proteinlevels (data not shown).

Experiment 7 Animal Model #1: Learned Helplessness Induced byInescapable Shock

The learned helplessness procedure consists of two separate stresssessions. On day 1, animals are placed in sound-attenuated operant boxes(Med Associates, St. Albans, Vt.) where they receive inescapable shock,with lights off and no levers present. Shock is delivered as a scrambledpulsed 0.8 mA current through metal floor bars. Both shock duration andintertrial intervals are randomly varied (1.5-60 sec and 1-30 sec,respectively), for a total shock exposure of 25 min. On day 2 rats areplaced in the operant boxes, and given exactly 15 trials of escapableshock, each lasting for a maximum 60 sec, with a fixed intertrialinterval of 24 seconds. Initiation of shock (0.8 mA) is accompanied bythe onset of a red cue light placed directly above a lever which whenpressed terminates the shock and turns off the cue light. A houselightoutside the immediate chamber is kept on during the entire trial. A barpress within the first 20 seconds of shock initiation is recorded as anescape response. A response between 20-60 seconds is classified asfailure to escape. After 60 seconds the shock is automaticallyterminated and the trial counted as a failure. Escape performance andlatency to escape are recorded for each animal over the 15 trials.Animals are classified as learned helpless (LH) if they fail to escapein 10 or more of the 15 trials. Rats that fail to escape in 5 or less ofthe 15 trials are termed resistant or non-learned helpless (nLH)⁸⁶⁻⁸⁷.Animals that fail 5-10 times are considered borderline. In addition tohome cage controls, another group may be placed in the boxes in day 1without receiving any shock. On day 2 this group receives escapableshock. It serves as a behavioral control only, by demonstrating thatcontrol rats can learn to escape during the 15 trials. For eachtreatment condition, 8 LH, 8 nLH and 8 cage controls are sacrificed 24hr after the escapable shock session via decapitation. Brains arequickly dissected and frozen on dry ice. All samples are kept at −80° C.for biochemistry and pharmacology analysis.

Experiment 8 Animal Model #2: Chronic Mild Stress

Rats are first trained to drink a 1% sucrose solution, by exposing themto sucrose in place of water for 48 h. They then receive a series ofsucrose preference tests, preceded by 23 h food and water deprivation,where each animal is presented simultaneously with 2 bottles, onecontaining 1% sucrose the other water. The position of the 2 bottles(right/left) is varied randomly from trial to trial and, within eachtrial, is counterbalanced across the animals in each group. During thetest, both bottles are removed after 30 min for weighing, and replacedby a second pair of preweighed bottles (with the positions of the 2bottles reversed), which are removed and weighed at 60 min. Tests aretimetabled at the start of the dark cycle (1800-1900 h) for half of theanimals and in the first half of the light cycle (1000-1100 h) for theother half. Following the final baseline test, each group of animalswill be divided into 2 subgroups, matched on the basis of their total(60-min) sucrose intake in the final baseline test. One pair ofsubgroups will be exposed to CMS for 6 weeks; the control subgroups willnot be stressed, other than the food/water deprivation that precedeseach sucrose preference test. In each of the first four weeks, the CMSschedule (adopted from ref 88) will consist of the following elements:Tues.1900-Wed.1000 h Paired housing (new partner); Wed.1000-1800 hStroboscopic illumination (in dark); Wed.1800-Thurs.1000 h Fooddeprivation in soiled cage (water in sawdust); Thurs.1000-1800 h 45°cage tilt; Thurs.1800-Fri.1000 h Mouse cage; Fri.1000-1800 h Pairedhousing (new partner); Fri.1800-Sat.1000 h Water deprivation;Sat.1000-1800 h Stroboscopic illumination (in dark); Sat.1800-Sun.0600 hLight on; Sun.0600-1800 h Intermittent lighting (off/on every 2 h);Mon.1100 or 1900 h 23-h food and water deprivation; Tue.1000 or 1800 h1-h sucrose intake test. In the final two weeks, the CMS timetable willbe rearranged, with paired housing (Mon. night) and food/waterdeprivation (Tues.night) immediately before sucrose intake test. Bodyweight will be monitored daily. After the last sucrose preference test,animals will be left undisturbed until next morning during when theywill be decapitated.

Experiment 9 Increase in the D1-D2 Coupling in Rats Subjected toForced-Swim Tests (FST)

To investigate the possibility that the D1-D2 coupling plays a role inthe pathology of depression, we examined the D1-D2 coupling using D1Rantibody to co-IP the D2R from striata of rats subjected to FST. The FST(or behavioural despair) is a good model to test for the efficacy ofantidepressant drugs. As shown in FIG. 8A, Striata from rats sacrificed3 hours or 3 days after the last FST trial exhibited a significantincrease in the D1-D2 coupling compared to control rats. Furthermore,there was no change in D1R or D2R protein levels that could account forthe increase in the D1-D2 complex formation (FIG. 8B, C). Surprisingly,rats sacrificed 3 days after the last FST trial exhibited a largerincrease in the D1-D2 coupling compared to rats sacrificed 3 hours afterthe last FST trial and suggested long-term changes occur after FSTtrials.

Experiment 10 Disruption of the D1-D2 Protein Complex in SchizophrenicBrain Tissue

To identify the physiological relevance for the D1-D2 receptor complexformation and specifically the effect of the antipsychotic medication,we carried out co-immunoprecipitation experiments in a double-blindmanner with 60 post-mortem brain striatum samples from the StanleyFoundation, which includes 15 samples from each of the four groups:control, schizophrenia, bipolar and severe depression. The four groupswere matched by age, sex, race, postmortem interval, pH, side of brain,and mRNA quality by the brain bank. The same amount of protein from eachsample was co-immunoprecipitated with D2 receptor antibody. Theprecipitated proteins were divided equally into two groups andimmunoblotted with either D1 antibody or D2 antibody. Consistent withour hypothesis, the co-immunoprecipitated D1 by the D2 antibody wassignificantly decreased in the post-mortem brain samples from bothschizophrenia and bipolar patients compared to control group (FIG. 4).The levels of directly immunoprecipitated D2 were not significantlydifferent among the four groups (data not shown). Interestingly, all theschizophrenia samples and 12 out of 15 of bipolar samples were frompatients treated with antipsychotics, indicating that the observed D2-D1interaction deficit seen in schizophrenia patients may not be a primaryaspect of schizophrenia pathophysiology, it may actually reflect thepharmacological effects of antipsychotics/D2 antagonists. Thus, wefurther tested the D1-D2 protein complex formation in rats chronicallytreated with haloperidol, a clinical antipsychotic as well as a D2antagonist (By pump 0.25 mg/kg/day for 2 weeks, to get continuousclinical occupancy⁸⁹). As shown in FIG. 9, while the directimmunoprecipitated D2 receptors remain unchanged (top panel), the D1-D2receptor complex formation is significantly decreased in rat withchronic antipsychotic treatment (bottom panel), suggesting thatantagonizing D2 function disrupts D1-D2 receptor coupling.

Experiment 11 Interfering Protein Peptide that is Able to Disrupt theD1-D2 Coupling Exerts Antidepressant Effect in the Forced-Swim Test

The FST is a stress model commonly used to test for the efficacy ofantidepressant drugs. Thus we will examine the effect of the interferingTAT peptides on rats using the FST, developed by Porsolt andcolleagues¹⁴⁶. Phase 1 of the FST consists of a preconditioning periodin which rats are forced to swim in an enclosed container of water(height of container, 40 cm; diameter of container, 20 cm; depth ofwater, 13 cm; temperature, 25° C.) for 15 min without a way to escape.In this predicament, rats will respond by becoming immobile. Twenty-fourhours after being removed from the container, each rat will be returnedto the water for a 5 min test (phase 2), and the behavior will berecorded by a video camera from the side of the cylinder. Rat's behaviorwill be classified for each 5 seconds and assigned to differentcategories according to the standard described previously⁹⁰ (climbing,diving, swimming, immobility and latency to immobility) from videotapesby a trained observer who is blinded to the experimental conditions). Toexamine the potential antidepressant effect of the interfering peptide,TAT-D2L_(IL3-29-2) (5 pmol) and TAT alone peptide were given ICV threetimes similar to antidepressant drugs: 1 hr and 5 hrs after the pre-swimtrial (phase 1) respectively and 1 hour before the swim test (phase 2).As shown in FIG. 10, Intra-PFC administration of TAT-D2L_(IL3-29-2), butnot TAT alone, decreased the frequencies of rat immobility and increasedthe frequencies of rat swimming and climbing behaviors in a 5 minsforced swimming test. Each value is the mean±S.E.M. for a group of 6rats. Data were analyzed by one-way analysis of variance (ANOVA).

One or more currently preferred embodiments have been described by wayof example. It will be apparent to persons skilled in the art that anumber of variations and modifications can be made without departingfrom the scope of the invention as defined in the claims.

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What is claimed is:
 1. A method for modulating dopamine (DA) receptorfunction in a mammal in need of such treatment comprising administeringan effective amount of a nucleic acid expressing a polypeptide of up to140 amino acids and comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 andcomprising SEQ ID NO:2; wherein the polypeptide disrupts D1-D2 couplingin the mammal.
 2. The method of claim 1, wherein the polypeptidecomprises an amino acid sequence that is identical to a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO:
 3. 3. The method of claim 1, wherein the nucleic acid furtherencodes a protein transduction domain.
 4. The method of claim 1, whereinthe method is for reducing the likelihood of depression and/or treatmentof depression.
 5. The method of claim 1, wherein the amino acid sequencecomprises SEQ ID NO: 1, SEQ ID NO: 3, or both.